Project Summary/Abstract The overall objective of Project 3 is to identify the elements of immunological memory induced by natural DENV infection and vaccination that correlate with outcome upon subsequent DENV exposure. Immunological memory is important to vaccine efficacy. Many DENV infections occur in the context of pre-existing immunological memory, and the consequences can be either beneficial (reduced risk of infection and/or disease) or pathological (increased risk of infection and/or disease); defining the characteristics that distinguish these outcomes will advance dengue vaccine development and implementation. Previous studies by our group have begun to identify associations between immunological memory and clinical outcomes of DENV infection. The proposed studies will address gaps in knowledge regarding the mechanisms of these associations and the kinetics of DENV-specific immunological memory. We hypothesize that specific populations of memory T and B lymphocytes defined by specificity and phenotype determine the outcome of infection, and that changes over time in populations of memory lymphocytes correlate with the time-varying risk of DENV infection and disease. These hypotheses will be tested using blood samples from Thai cohort studies as well as extended observation of a unique cohort of participants in the phase III trial of the chimeric yellow fever-dengue vaccine in Cebu, the Philippines, who have had annual blood collections and active surveillance for acute dengue illnesses for ~5 years. The Specific Aims to be addressed in Project 3 are to: 1) define the memory T and B cell populations in pre-infection blood samples that are associated with the outcome of subsequent DENV infection; 2) define the changes in frequency, specificity, and function of DENV- specific T cell and B cell populations over time after natural infection and their associations with incident DENV infections; and 3) define the changes in frequency, specificity, and function of DENV-specific T cell and B cell populations over time after vaccination and their associations with risk of DENV infection and disease. Project 3?s focus on DENV-specific immunological memory and its correlations with clinical outcome directly address the overall objectives of this Program Project. Assays developed in Project 3 will be extended to the study cohorts of Projects 1 and 2, and vice versa. Project 3 will utilize Core A for project administration, Core B for data management and statistical analysis, and Core C for processing of clinical specimens, diagnostic testing, and assays of antibody specificity and function. The results of these studies will be useful to improve understanding of previous vaccine trial results and to guide the evaluation of current vaccine candidates.